U.S. patent application number 17/168465 was filed with the patent office on 2021-08-19 for cell purification device.
This patent application is currently assigned to SHIMADZU CORPORATION. The applicant listed for this patent is SHIMADZU CORPORATION. Invention is credited to Akihiro ISHIKAWA, Masayuki NISHIMURA.
Application Number | 20210255106 17/168465 |
Document ID | / |
Family ID | 1000005406423 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210255106 |
Kind Code |
A1 |
NISHIMURA; Masayuki ; et
al. |
August 19, 2021 |
CELL PURIFICATION DEVICE
Abstract
A device capable of targeting and selectively removing (killing)
an unnecessary cell that remains in a cell population induced to
differentiate from pluripotent stem cells and causes tumorigenesis
after transplantation, and automatically and efficiently collecting
an object cell. The device includes a holder configured to hold a
cell suspension containing cells and an antibody-photosensitizer
conjugate obtained by conjugating a targeting molecule that binds
to a surface protein of a cell to be removed and a fluorescently
labeled substance, a radiation light source configured to radiate
radiation to change a physical property of the fluorescently
labeled substance contained in the holder, a detector configured to
detect abundance of the cells in the holder, and a
control/processing unit configured to control the radiant light
source.
Inventors: |
NISHIMURA; Masayuki;
(Columbia, MD) ; ISHIKAWA; Akihiro; (Kyoto-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIMADZU CORPORATION |
Kyoto-shi |
|
JP |
|
|
Assignee: |
SHIMADZU CORPORATION
Kyoto-shi
JP
|
Family ID: |
1000005406423 |
Appl. No.: |
17/168465 |
Filed: |
February 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62977983 |
Feb 18, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 2300/0654 20130101; G01N 21/6486 20130101; B01L 2300/168
20130101; G01N 21/0303 20130101; G01N 21/6428 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; B01L 3/00 20060101 B01L003/00; G01N 21/03 20060101
G01N021/03 |
Claims
1. A purification device configured to purify cells, the
purification device comprising: a holder configured to hold a cell
suspension containing cells and an antibody-photosensitizer
conjugate obtained by conjugating a targeting molecule that binds
to a surface protein of a cell to be removed and a fluorescently
labeled substance; a radiation light source configured to radiate
radiation to change a physical property of the fluorescently
labeled substance contained in the holder; a detector configured to
detect abundance of the cells in the holder; and a
control/processing unit configured to control the radiant light
source; wherein the antibody-photosensitizer conjugate kills the
cell to be removed by a change of the physical property of the
fluorescently labeled substance; and the control/processing unit is
configured to determine whether or not the cell to be removed has
been killed based on the abundance of the cells detected by the
detector, and to continue irradiation with the radiation from the
radiation light source when the cell to be removed has not been
killed.
2. The purification device according to claim 1, wherein a
container containing the cell suspension containing the cells, a
conjugate storage configured to store the antibody-photosensitizer
conjugate, and the holder are connected by flow paths; and the flow
paths include pumps configured to introduce the cell suspension and
the antibody-photosensitizer conjugate into the holder.
3. The purification device according to claim 1, wherein the cells
include one or both of cultured cells and isolated cells.
4. The purification device according to claim 1, wherein the
detector includes a fluorescence detector configured to detect
fluorescence emitted from the fluorescently labeled substance, or a
cell imager.
5. The purification device according to claim 1, wherein the
control/processing unit is configured to radiate radiation having
enough energy to excite the fluorescently labeled substance but not
kill the cell to be removed before killing the cell to be removed
by radiating radiation to change the physical property of the
fluorescently labeled substance, and to detect a position of the
cell to be removed in the holder and then irradiate the position
with radiation having enough energy to kill the cell to be
removed.
6. The purification device according to claim 1, wherein the
control/processing unit is configured to determine whether or not
the cell to be removed has died based on a presence of fluorescence
detected by radiating radiation having enough energy to excite the
fluorescently labeled substance but not kill the cell to be removed
after radiating radiation having enough energy to kill the cell to
be removed.
7. The purification device according to claim 1, wherein the holder
includes a cell container containing the cell suspension containing
the cells.
8. The purification device according to claim 1, further
comprising: a liquid feeding pump configured to flow the cell
suspension; wherein the holder forms a flow path including an inlet
for taking in the cell suspension, an outlet for discharging the
cell suspension containing purified cells, and the liquid feeding
pump; and the control/processing unit is configured to drive the
liquid feeding pump to discharge the cell suspension in the flow
path from the outlet when determining that removal of the cell to
be removed has been completed based on the abundance of the cells
detected by the detector.
9. The purification device according to claim 8, further
comprising: a cell container configured to house cells; wherein the
flow path forms a circulation flow path; the cell container is
provided in a return flow path of the circulation flow path; and
the control/processing unit is configured to drive the liquid
feeding pump to feed the cell suspension in the flow path to the
cell container when determining that the cell to be removed has
died based on detected fluorescence abundance.
10. The purification device according to claim 9, comprising: an
analyzer configured to analyze the cell suspension containing the
antibody-photosensitizer conjugate and a ligand of the
antibody-photosensitizer conjugate; a flow path configured to
supply the cell suspension to the analyzer; and a switching valve
configured to selectively switch any flow path to the analyzer or
the cell container; wherein the switching valve is provided in the
return flow path of the circulation flow path.
11. The purification device according to claim 10, wherein the
analyzer includes a liquid chromatograph mass spectrometer.
12. The purification device according to claim 1, wherein the
radiation radiated by the radiation light source has a wavelength
of 660 to 740 nm.
13. The purification device according to claim 1, wherein the
radiation radiated by the radiation light source includes
near-infrared light; and the fluorescently labeled substance
activated by irradiation with the near-infrared light includes a
phthalocyanine dye.
14. The purification device according to claim 13, wherein the
phthalocyanine dye is IRDye.RTM. 700.
15. The purification device according to claim 1, wherein a ligand
of the antibody-photosensitizer conjugate is a C14H33NO10S3Si
fragment.
Description
DETAILED DESCRIPTION OF THE INVENTION
Technical Field
[0001] The present invention relates to a device configured to kill
a specific cell among cells cultured on a cell culture vessel and
to extract an object cell, and a device such as a light source
device or a cell culture vessel used to conduct that, for
example.
BACKGROUND ART
[0002] In recent years, research and development of regenerative
medicine technology and drug discovery using somatic stem cells,
embryonic stem cells, and induced pluripotent stem cells have been
actively carried out. In this type of research and development, it
is extremely important to be able to efficiently mass-produce
object cells and tissues.
[0003] When cell culture is performed, it is common to perform
subculture by cutting out a portion of a cell aggregate (colony)
proliferated in a culture medium, replanting it in a new culture
medium, and culturing it again when the cell aggregate (colony)
grown in the culture medium satisfies a predetermined seeding
density or area value, for example.
[0004] At present, subculture of the proliferated cells relies
exclusively on manual operation, but this requires time and effort,
and may cause irregularities in the sizes of the cut-out cells,
which may result in variations in the state of growth of the
subcultured cells.
[0005] Cases have been reported in which undifferentiated cells
remain in a differentiated cell population and cause tumorigenesis
(teratoma, carcinogenesis) when pluripotent stem cells such as ES
cells or iPS cells are cultured under conditions for
differentiating them into cells such as myocardium or nerves
(Non-Patent Document 1). Furthermore, iPS cells are artificially
reprogrammed cells, and thus there are safety issues such as the
risk of tumorigenesis due to introduction of proto-oncogenes such
as c-Myc or use of viral vectors, and the risk of tumorigenesis due
to resistance to differentiation depending on the type of deriving
somatic cells to be the derivation.
[0006] Even in the elucidation of pathological conditions using
disease-specific iPS cells derived from patients and the
development of drug discovery for the disease, contamination of
undifferentiated cells other than the object differentiated cells
causes other cell types, and thus accurate experiments cannot be
performed due to mixing of cells other than the object cells.
Similarly, in the application of human ES cells and iPS cells in
terms of supplying normal human cells to drug efficacy and toxicity
tests in drug discovery, contamination of undifferentiated cells
other than the target differentiated cells causes other cell types,
and thus it is conceivable that mixing of cells other than the
target cells reduces the accuracy of measurement and the
reliability thereof.
[0007] Therefore, when cells that have failed in differentiation
into specific tissues or organs or have not differentiated or
unnecessary cells such as tumorigenic cells are detected, as a
countermeasure, the culture vessel containing these unnecessary
cells may be discarded. However, it leads to a decrease in the
recovery rate of normally differentiated object cells or tissues,
and increases the cost of regenerative medicine.
[0008] Thus, it is desirable to improve the recovery rate of object
cells or tissues without wasting the remaining cells by killing or
removing the unnecessary cells existing in the culture vessel.
[0009] As a method for selectively killing unnecessary cells in a
culture vessel, a method for radiating active energy rays such as
visible light, ultraviolet rays, infrared rays, or radiation has
been proposed (Patent Document 1). That is, a photo-acid-generating
agent that generates an acidic substance by being irradiated with
active energy rays such as visible light, ultraviolet rays,
infrared rays, or radiation is applied to a surface of the culture
vessel in advance, and the active energy rays are radiated for
about 10 seconds to 10 minutes to a portion in which cells to be
killed among the cells cultured in this culture vessel exist such
that the acidic substance is generated to kill the target cells. A
digital micromirror device (DMD), a liquid crystal shutter array,
an optical spatial modulation element, a photomask, or the like is
used to control a region to be irradiated with the active energy
rays.
[0010] The method disclosed in Patent Document 1 requires a long
time to radiate the active energy rays in order to kill the target
cells, and it can be said that there is still room for improvement
for the upcoming mass production of regenerative medicine cells. In
addition, in a micro-projection system using a DMD or the like,
most of energy supplied from an active energy source (light source)
is wasted. Moreover, it is difficult to keep the intensity
distribution of the active energy rays radiated to the
photo-acid-generating agent uniform.
[0011] It is also conceivable to directly irradiate the unnecessary
cells with the active energy rays such as high-energy pulse lasers
in order to speed up a process of killing the unnecessary cells.
However, the active energy rays to be radiated need to hit the cell
nuclei, and the cells cannot be surely killed unless the target
cells are irradiated a plurality of times. Furthermore, there is an
essential problem that the thermal effect on the object cells
around the unnecessary cells that are directly irradiated with the
active energy rays is unavoidable.
PRIOR ART
Patent Document
[0012] [Patent Document 1] International Publication No.
2011/125615
Non-Patent Document
[0012] [0013] [Non-Patent Document 1] Miura, K. et al., Nat.
Biotechnol. 27: 743-745 (2009)
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0014] The present invention aims to provide a device capable of
targeting and selectively removing (killing) unnecessary cells
remaining in a cell population induced to differentiate from
specific cells and including cells that have failed to
differentiate or have not differentiated into desired tissues or
organs, tumorigenic cells, etc., and automatically and efficiently
collecting an object cell.
Means for Solving the Problem
[0015] The present invention aims to provide a device using
photoimmunotherapy that enables targeted killing of a specific cell
that has bound to an antibody-IR700 conjugate by activating IR700
by irradiation with near-infrared light, focusing on an
antibody-photosensitizer conjugate (antibody-IR700 conjugate)
obtained by conjugating an antibody or another targeting molecule
that targets a cell surface protein with a chemical substance that
is a water-soluble phthalocyanine derivative called IR700.
[0016] Therefore, the present invention is:
[0017] a purification device configured to purify cells, the
purification device including:
[0018] a holder configured to hold a cell suspension containing
cells and an antibody-photosensitizer conjugate obtained by
conjugating a targeting molecule that binds to a surface protein of
a cell to be removed and a fluorescently labeled substance;
[0019] a radiation light source configured to radiate radiation to
change a physical property of the fluorescently labeled substance
contained in the holder;
[0020] a detector configured to detect abundance of the cells in
the holder; and
[0021] a control/processing unit configured to control the radiant
light source; wherein
[0022] the antibody-photosensitizer conjugate kills the cell to be
removed by a change of the physical property of the fluorescently
labeled substance; and
[0023] the control/processing unit is configured to determine
whether or not the cell to be removed has been killed based on the
abundance of the cells detected by the detector, and to continue
irradiation with the radiation from the radiation light source when
the cell to be removed has not been killed;
[0024] the purification device wherein
[0025] a container containing the cell suspension containing the
cells, a conjugate storage configured to store the
antibody-photosensitizer conjugate, and the holder are connected by
flow paths; and
[0026] the flow paths include pumps configured to introduce the
cell suspension and the antibody-photosensitizer conjugate into the
holder;
[0027] the purification device, wherein the control/processing unit
is configured to radiate radiation having enough energy to excite
the fluorescently labeled substance but not kill the cell to be
removed before killing the cell to be removed by radiating
radiation to change the physical property of the fluorescently
labeled substance, and to detect a position of the cell to be
removed in the holder and then irradiate the position with
radiation having enough energy to kill the cell to be removed;
[0028] the purification device, wherein the control/processing unit
is configured to determine whether or not the cell to be removed
has died based on a presence of fluorescence detected by radiating
radiation having enough energy to excite the fluorescently labeled
substance but not kill the cell to be removed after radiating
radiation having enough energy to kill the cell to be removed;
[0029] the purification device, wherein the holder includes a cell
container containing the cell suspension containing the cells;
[0030] the purification device further including:
[0031] a liquid feeding pump configured to flow the cell
suspension; wherein
[0032] the holder forms a flow path including an inlet for taking
in the cell suspension, an outlet for discharging the cell
suspension containing purified cells, and the liquid feeding pump;
and
[0033] the control/processing unit is configured to drive the
liquid feeding pump to discharge the cell suspension in the flow
path from the outlet when determining that removal of the cell to
be removed has been completed based on the abundance of the cells
detected by the detector;
[0034] the purification device further including:
[0035] a cell incubator configured to culture cells; wherein
[0036] the flow path forms a circulation flow path;
[0037] the cell incubator is provided in a return flow path of the
circulation flow path; and
[0038] the control/processing unit is configured to drive the
liquid feeding pump to feed the cell suspension in the flow path to
the cell incubator when determining that the cell to be removed has
died based on detected fluorescence abundance;
[0039] the purification device including:
[0040] an analyzer configured to analyze the cell suspension
containing the antibody-photosensitizer conjugate and a ligand of
the antibody-photosensitizer conjugate;
[0041] a flow path configured to supply the cell suspension to the
analyzer; and
[0042] a switching valve configured to selectively switch any flow
path to the analyzer or the cell incubator; wherein
[0043] the switching valve is provided in the return flow path of
the circulation flow path;
[0044] the purification device, wherein the analyzer includes a
liquid chromatograph mass spectrometer;
[0045] the purification device, wherein the radiation radiated by
the radiation light source has a wavelength of 660 to 740 nm;
[0046] the purification device, wherein
[0047] the radiation radiated by the radiation light source
includes near-infrared light; and
[0048] the fluorescently labeled substance activated by irradiation
with the near-infrared light includes a phthalocyanine dye;
[0049] the purification device, wherein the phthalocyanine dye is
IRDye.RTM. 700; and
[0050] the purification device, wherein a ligand of the
antibody-photosensitizer conjugate is a C14H33NO10S3Si
fragment.
[0051] Note that the cells in the present invention include not
only cultured cells but also isolated cells such as pancreatic
islets.
[0052] The detector includes a fluorescence detector configured to
detect fluorescence emitted from the fluorescently labeled
substance, or a cell imager.
[0053] A conventionally known mechanism of photoimmunotherapy is as
follows:
[0054] Photoimmunotherapy (PIT), which is a new cancer treatment
method discovered by Senior Researcher Hisataka Kobayashi et al. of
the National Cancer Institute, is a therapeutic method that uses,
as a drug, an antibody-photosensitizer conjugate (antibody-IR700
conjugate) to which a chemical substance that is a water-soluble
phthalocyanine derivative called IR700 binds, and has extremely few
side effects because it is not toxic to anything other than cancer
cells (for example, U.S. Patent Application Publication Nos.
2017/0122853, 2016/001589, 2014/0120199, 2013/0336995, 2012/0010558
(Kobayashi et al.), and Non-Patent Document "Kazuhide Sato et al.,
ACS Cent. Sci. 2018, 4, 1559-1569"). Antibodies that are used in
PIT and target cancerous cells and light absorbers/photosensitizers
of phthalocyanine dye such as IRDye.RTM. 700 form
antibody-photosensitizer conjugates. When these
antibody-photosensitizer conjugates are injected into a patient and
pass through the bloodstream to reach a tumor containing cancer
cells, they leak from permeable blood vessels near the tumor and
then bind to antigens, cell surface proteins that are specifically
present in cancer cells.
[0055] After the antibody-photosensitizer conjugates bind to the
cancer cells, near-infrared light is radiated to the cancer cells
from either inside or outside the body. The near-infrared light is
highly permeable to living organisms, and thus it is transmitted
without causing damage to living tissues and the like. Irradiation
with this near-infrared light activates IRDye.RTM. 700 and induces
an axial ligand (C14H33NO10S3Si fragment) release reaction such
that the shapes of the antibodies and antigens physically change,
and physical stresses within cell membranes that lead to an
increase in transmembrane water are induced. When water enters the
cancer cells, they swell, and the internal cell pressure rises such
that the cancer cells rupture and die. When the cancer cells
rupture, internal substances inside the cancer cells are released
into extracellular spaces. At that time, a body's immune system
detects the internal substances and cell debris as "foreign
substances", and thus an immune response that promotes cancer
destruction is activated. Specifically, T-lymphocytes, which are a
type of white blood cell, and thymocytes ("T cells") attack and
destroy the cell debris. FIG. 4 shows a mechanism of cancer cell
death.
[0056] Therefore, the "cell to be removed" in the present invention
refers to any cell that has become a tumor, and can include cancer
cells, for example. Specific examples include squamous cell
carcinoma, but are not limited to this.
[0057] A protein or peptide that is a targeting molecule that binds
to the surface protein (marker molecule) of the cell to be removed
varies depending on the type of marker molecule, and examples
thereof include cetuximab that binds to an epidermal growth factor
receptor (EGFR). However, the present invention is not limited to
cetuximab, but any antibody that binds to a marker molecule that is
more strongly expressed than normal cells may be used. For example,
panitumumab, zalutumumab, nimotuzumab, tositumomab, rituximab,
ibritumomab tiuxetan, daclizumab, gemtuzumab, alemtuzumab, a
CEA-scan Fab fragment, OC125, ab75705, B72.3, bevacizumab,
basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475,
BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321,
BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166,
dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469,
MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C,
rHIgM12B7, ulocuplumab, BKT140, varlilumab, ARGX-110, MGA271,
lirilumab, IPH2201, AGX-115, emactuzumab, CC-90002, MNRP1685A, or
an antigen-binding fragment thereof, which is described in Japanese
Translation of PCT International Application Publication Nos.
2014-523907 and 2018-528268, may be selected.
[0058] The protein or peptide of the present invention can be
produced by a known (poly) peptide synthesis method. The peptide
synthesis method may be either a solid-phase synthesis method or a
liquid-phase synthesis method, for example. The target peptide can
be produced by condensing a partial peptide that can constitute the
peptide of the present invention or amino acid with a residual
portion, and removing a protecting group when the product has the
protecting group.
[0059] The peptide obtained in this manner can be purified and
isolated by a known purification method. Examples of the
purification method include solvent extraction, distillation,
column chromatography, liquid chromatography, recrystallization,
and combinations thereof.
[0060] When the peptide obtained by the above method is in a free
form, the form can be converted to a suitable salt by a known
method or a method analogous thereto. Conversely, when the peptide
is obtained in the form of a salt, the salt can be converted to a
free form or another salt by a known method or a method analogous
thereto.
[0061] The mode of binding of the protein or peptide of the present
invention with one or more components is not particularly limited.
The binding may be direct or indirect via a linker etc. The binding
may be by covalent binding, non-covalent binding, or a combination
thereof. One or more components may be directly or indirectly
bonded at an N-terminal, a C-terminal, or another position of the
peptide of the present invention. The binding of a peptide with
another component (or second peptide) is well known in the art, and
the binding may be by any known means in the conjugate of the
present invention.
[0062] For example, when the binding is via a linker, known
crosslinkers such as NHS ester, imide ester, maleimide,
carbodiimide, allyl azide, diazirine, isocyanide, psoralen, etc.
can be used. Depending on the crosslinker to be used, the peptide
of the present invention may be modified as appropriate. For
example, a cysteine can be added in advance to the C-terminal of
the peptide of the present invention for binding with a maleimide
linker.
[0063] A fluorescently labeled substance is conjugated to the above
protein or peptide. The fluorescently labeled substance may be any
substance that is activated (changes its physical properties) by
irradiation with radiation, electromagnetic waves, or sound waves,
for example, and emits fluorescence. The radiation includes
radiation in a narrow sense, i.e., particle radiation such as beta
rays, neutron rays, proton beams, heavy-ion beams, meson beams,
etc., and electromagnetic radiation such as gamma rays and X-rays.
The electromagnetic waves include so-called light rays such as
infrared rays, visible rays, and ultraviolet rays, and radio waves,
and the sound waves also include ultrasonic waves.
[0064] In a mechanism of cell membrane destruction according to the
present invention, a drug becomes hydrophobic due to removal of a
hydrophilic group called a ligand, for example, of the
fluorescently labeled substance, and a failure occurs in the cell
membrane, unlike the conventionally known photodynamic therapy
(PDT). That is, in the present invention, the physical properties
of the fluorescently labeled substance change in a state in which a
fluorescently labeled substance-peptide conjugate binds to the
marker molecule (on the cell membrane) that exists on a surface of
the cell to be removed, and membrane-conjugate deformation or
aggregate formation damage a cancer cell membrane.
[0065] In the present invention, a change of the physical
properties of the fluorescently labeled substance becomes a "death
switch", and this switch can be turned on by a remote control of
light that is not toxic to a living body, such as near-infrared
light. It is a completely new cell killing method that can turn
only the conjugate binding to a cancer cell into poison by
light.
[0066] In the present invention, any substance that has the
aforementioned "death switch" property and emits fluorescence can
be used. However, a preferable fluorescently labeled substance is a
photosensitive compound.
[0067] Phthalocyanine dye can be mentioned as a more preferable
fluorescently labeled substance used in the present invention.
Phthalocyanines are a group of photosensitizer compounds having a
phthalocyanine ring system. Phthalocyanines are azaporphyrins that
contain four benzoindole groups connected by nitrogen bridges in a
16-membered ring of alternating carbon and nitrogen atoms (i.e.,
C32H16N8) which form stable chelates with metal and non-metal
cations. In these compounds, the ring center is occupied by a metal
ion (either a diamagnetic or a paramagnetic ion) that may,
depending on the ion, carry one or two ligands. In addition, the
ring periphery may be either unsubstituted or substituted.
[0068] Phthalocyanines strongly absorb red or near infrared rays
with absorption peaks falling between about 600 nm and 810 nm,
which, in some cases, allow deep penetration of tissue by the
light. Phthalocyanines are generally photostable. This photo
stability is typically advantageous in pigments and dyes and in
many of the other applications of phthalocyanines. The
phthalocyanine dye has a maximum light absorption in the near
infrared (NIR range). In some embodiments, the phthalocyanine dye
has a maximum light absorption wavelength between 400 nm and 900
nm, such as between 600 nm and 850 nm or between 680 nm and 850 nm,
for example at approximately 690.+-.50 nm or 690.+-.20 nm. In some
embodiments, the phthalocyanine dye can be excited efficiently by
commercially available laser diodes that emit light at these
wavelengths.
[0069] In some embodiments, the phthalocyanine dye containing a
reactive group is IR700 NHS ester, such as IRDye 700DX NHS ester
(LiCor 929-70010, 929-70011).
[0070] Means for changing the physical properties of the
fluorescently labeled substance can be irradiation with radiation,
electromagnetic waves, or sound waves, for example, but is not
limited thereto. It can also be done by chemical means. In the case
of irradiation, irradiation with therapeutic doses of radiation or
electromagnetic waves at a wavelength in the range of 400 nm to
about 900 nm or about 400 nm to about 900 nm, 500 nm to about 900
nm or about 500 nm to about 900 nm, 600 nm to about 850 nm or about
600 nm to about 850 nm, 600 nm to about 740 nm or about 600 nm to
about 740 nm, about 660 nm to about 740 nm, about 660 nm to about
710 nm, about 660 nm to about 700 nm, about 670 nm to about 690 nm,
about 680 nm to about 740 nm, or about 690 nm to about 710 nm, for
example, is performed. In some embodiments, cells, such as tumors,
are irradiated with therapeutic doses of radiation or
electromagnetic waves at a wavelength from 600 nm to 850 nm, such
as 660 nm to 740 nm. In some embodiments, cells, such as tumors,
are irradiated at a wavelength of at least 600 nm, 620 nm, 640 nm,
660 nm, 680 nm, 700 nm, 720 nm, or 740 nm, or at least about 600
nm, about 620 nm, about 640 nm, about 660 nm, about 680 nm, about
700 nm, about 720 nm, or about 740 nm, such as 690.+-.50 nm or
about 680 nm, for example.
[0071] In some embodiments, cells, such as tumors, are irradiated
with a dose of at least 1 J/cm.sup.2, such as at least 10
J/cm.sup.2, at least 30 J/cm.sup.2, at least 50 J/cm.sup.2, at
least 100 J/cm.sup.2, or at least 500 J/cm.sup.2. In some
embodiments, the dose of irradiation is 1 to about 1000 or about 1
to about 1000 J/cm.sup.2, about 1 to about 500 J/cm.sup.2, about 5
to about 200 J/cm.sup.2, about 10 to about 100 J/cm.sup.2, or about
10 to about 50 J/cm.sup.2. In some embodiments, cells, such as
tumors, are irradiated with a dose of at least 2 J/m.sup.2, 5
J/cm.sup.2, 10 J/cm.sup.2, 25 J/cm.sup.2, 50 J/cm.sup.2, 75
J/cm.sup.2, 100 J/cm.sup.2, 150 J/cm.sup.2, 200 J/cm.sup.2, 300
J/cm.sup.2, 400 J/cm.sup.2, or 500 J/cm.sup.2, or at least about 2
J/cm.sup.2, 5 J/cm.sup.2, 10 J/cm.sup.2, 25 J/cm.sup.2, 50
J/cm.sup.2, 75 J/cm.sup.2, 100 J/cm.sup.2, 150 J/cm.sup.2, 200
J/cm.sup.2, 300 J/cm.sup.2, 400 J/cm.sup.2, or 500 J/cm.sup.2.
[0072] In some embodiments, cells, such as tumors, are irradiated
or illuminated with a dose of at least 1 J/fiber length cm, such as
at least 10 J/fiber length cm, at least 50 J/fiber length cm, at
least 100 J/fiber length cm, at least 250 J/fiber length cm, or at
least 500 J/fiber length cm. In some embodiments, the dose of
irradiation is 1 to about 1000 or about 1 to about 1000 J/fiber
length cm, about 1 to about 500 J/fiber length cm, about 2 to about
500 J/fiber length cm, about 50 to about 300 J/fiber length cm,
about 10 to about 100 J/fiber length cm, or about 10 to about 50
J/fiber length cm. In some embodiments, cells, such as tumors, are
irradiated with a dose of at least 2 J/fiber length cm, 5 J/fiber
length cm, 10 J/fiber length cm, 25 J/fiber length cm, 50 J/fiber
length cm, 75 J/fiber length cm, 100 J/fiber length cm, 150 J/fiber
length cm, 200 J/fiber length cm, 250 J/fiber length cm, 300
J/fiber length cm, 400 J/fiber length cm or 500 J/fiber length cm,
or at least about 2 J/fiber length cm, 5 J/fiber length cm, 10
J/fiber length cm, 25 J/fiber length cm, 50 J/fiber length cm, 75
J/fiber length cm, 100 J/fiber length cm, 150 J/fiber length cm,
200 J/fiber length cm, 250 J/fiber length cm, 300 J/fiber length
cm, 400 J/fiber length cm or 500 J/fiber length cm.
[0073] In some embodiments, the dose of irradiation or illumination
in a human subject is 1 to about 400 J/cm.sup.2 or about 1 to about
400 J/cm.sup.2, about 2 to about 400 J/cm.sup.2, about 1 to about
300 J/cm.sup.2, about 10 to about 100 J/cm.sup.2, or about 10 to
about 50 J/cm.sup.2, e.g., at least 10 J/cm.sup.2 or at least about
10 J/cm.sup.2, 10 J/cm.sup.2, within 10 J/cm.sup.2 or within about
10 J/cm.sup.2, 10 J/cm.sup.2 or about 10 J/cm.sup.2, at least 30
J/cm.sup.2, at least 50 J/cm.sup.2, or at least 100 J/cm.sup.2. In
some embodiments, the dose of irradiation in a human subject is 1
to 300 J/fiber length cm or about 1 to 300 J/fiber length cm, 10 to
100 J/fiber length cm or about 10 to 100 J/fiber length cm, or 10
to 50 J/fiber length cm or about 10 to 50 J/fiber length cm, e.g.,
at least 10 J/fiber length cm or at least about 10 J/fiber length
cm, 10 J/fiber length cm, within 10 J/fiber length cm or within
about 10 J/fiber length cm, 10 J/fiber length cm or about 10
J/fiber length cm, at least 30 J/fiber length cm, at least 50
J/fiber length cm, or at least 100 J/fiber length cm. In some
cases, the dose of irradiation in human subjects for achieving PIT
is less than the dose required for PIT in mice.
Effect of the Invention
[0074] According to the present invention, among the cultured or
isolated cells, the cell to be removed can be selectively removed
(killed) non-invasively without damaging the object cell by using
the antibody-photosensitizer conjugate and radiation, and thus it
is possible to efficiently collect only the object cell.
[0075] Furthermore, it is possible to prevent omission of removal
of the cell to be removed and omission of washing of the
antibody-photosensitizer conjugate in the cell suspension, and thus
the object cell can be normally cultured even after cell
purification. In addition, the risk of tumorigenesis of cells
transplanted into the patient can be greatly reduced, and thus
contribution to the development of regenerative medicine and the
development of effective treatment methods for diseases can be
expected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1 A diagram of an embodiment of a flow-through
purification device of the present invention.
[0077] FIG. 2 A flowchart showing the control contents of a
control/processing unit.
[0078] FIG. 3 A diagram of an embodiment of a batch purification
device of the present invention.
[0079] FIG. 4 A diagram showing a mechanism of cancer cell
death.
MODES FOR CARRYING OUT THE INVENTION
Embodiment 1: Flow-Through
[0080] A flow-through purification device and a cell purification
method using the device, which are an embodiment of the present
invention, are described with reference to the accompanying
drawings. FIG. 1 is a block diagram showing the configuration of a
purification device of this embodiment.
[0081] As shown in FIG. 1, the purification device includes 1:
holder, 2: detector, 3: radiation light source, 4: cell incubator,
5: liquid feeding pump, 6: introduction pump, 7: conjugate storage,
8: switching valve, 9: analyzer, and 10: control/processing unit.
The control/processing unit 10 controls the energy intensity of
radiation by the radiation light source 3, the amount of cell
suspension fed by the liquid feeding pump 6, adjustment of the
switching valve 8, analysis execution in the analyzer 9, etc. The
fluorescence intensity detected by the detector 2 and analysis data
obtained by the analyzer 9 are input to the control/processing unit
10. In general, at least some of the functions of the
control/processing unit 10 can be performed by using a personal
computer as a hardware resource and running dedicated
control/processing software pre-installed on the personal computer
on the computer.
[0082] The cell incubator 4 and the holder 1 are connected to each
other by flow paths via the liquid feeding pump 6 and the switching
valve 8. Furthermore, the analyzer 9 is connected to the holder 1
by a flow path by switching the switching valve 8. The cell
incubator 4 may be detachable from these flow paths.
[0083] The analyzer 9 may analyze a cell suspension or only a
culture medium of the cell suspension. When only the culture medium
is analyzed, a filter (not shown) for collecting cells from the
cell suspension is provided at any position in the flow path that
connects the holder 1 to the switching valve 8 when the switching
valve 8 is connected to the analyzer 9. The filter is manually or
automatically arranged. Isopore Membrane Filter manufactured by
Milipore (HTTP pore diameter: 0.4 .mu.m, diameter: 47 mm, 047 00)
can be used, for example, but the filter is not limited to this.
The cells collected by the filter may be manually seeded in the
cell incubator 4, or after the switching valve 8 is connected to
the cell incubator 4, the cells may be automatically fed to the
cell incubator 4 together with the cell suspension.
[0084] When the cells are subcultured in the cell incubator 4, they
may be subcultured manually or automatically. As the culture medium
used to culture the cells in the cell incubator 4, a culture medium
commonly used to culture stem cells, e.g. DMEM/F12 or a culture
medium containing DMEM/F12 as a main component (such as mTeSR1 or
TeSR-E8), can be used when a target to be cultured is stem cells
such as ES cells or iPS cells, but the culture medium is not
limited to this.
[0085] A purification procedure for purifying the cells using the
purification device described above and a control by the
control/processing unit 10 to perform the purification are now
described based on a flowchart shown in FIG. 2.
[0086] A cell suspension contained in the cell incubator 4 and
containing a culture medium and cells is fed to the holder 1 by the
liquid feeding pump 6 (step 1). Upon completion of feeding of the
cell suspension to the holder 1 or during the feeding,
antibody-photosensitizer conjugates are delivered from the
conjugate storage 7 by the introduction pump 6 (step 2). Then, the
holder 1 is irradiated with radiation having enough energy to
excite fluorescently labeled substances in the
antibody-photosensitizer conjugates but not kill cells to be
removed, and the positions of the cells to be removed in the holder
are detected (step 3). The energy intensity of the radiation may be
controlled by adjusting the irradiation time or irradiation
diameter, for example, by the control/processing unit 10. Regarding
the existence of the cells to be removed, for example, the cells to
be removed may be detected by introducing the
antibody-photosensitizer conjugates into cells known to be normal
cells, storing the fluorescence intensity detected when radiation
is radiated, comparing it with the fluorescence intensity detected
in step 3, and making a determination based on whether or not the
cells are to be removed.
[0087] Regarding the locations of the cells to be removed,
positions assumed to be the cells to be removed may be detected on
image data by capturing an observation image of the holder 1 in an
imaging unit (not shown) such as a CCD camera via a reflecting
mirror (not shown) and a condenser lens (not shown), inputting
image data corresponding to the observation image reproduced in the
imaging unit to the control/processing unit 10, introducing the
antibody-photosensitizer conjugates into cells known to be normal
cells, and comparing the fluorescence intensity detected when
radiation is radiated with the fluorescence intensity detected in
step 3. The control/processing unit 10 may control the radiation
light source 4 to irradiate the detected locations of the cells to
be removed with radiation. At that time, the radiation light source
4 may be moved to the irradiation position by a drive (not
shown).
[0088] Furthermore, the image data may be displayed by a display
(not shown).
[0089] Then, the positions of the cells to be removed detected in
step 4 are irradiated with radiation having enough energy to kill
the cells to be removed (step 4). Then, in order to detect whether
or not the cells to be removed remain in the holder 1, radiation
having enough energy to excite the fluorescently labeled substances
in the antibody-photosensitizer conjugates but not kill the cells
to be removed is radiated (step 5). When it is confirmed that the
cells to be removed remain, the process operation in step 4 is
performed again. When it is determined in step 5 that the cells to
be removed have died in the holder 1, the antibody-photosensitizer
conjugates and the ligands of the antibody-photosensitizer
conjugates contained in the cell suspension are removed (step 6).
Examples of a method for removing the antibody-photosensitizer
conjugates and the ligands of the antibody-photosensitizer
conjugates include a method for pellet collection or collection by
a filter. After the cells to be removed have died, an object cell
that remains in the cell suspension may be further washed with a
serum-free medium, phosphate buffered saline, culture medium, or
blocking buffer. Examples of the serum-free medium include EMEM and
RPMI. Next, in order to determine whether or not the
antibody-photosensitizer conjugates and the ligands of the
antibody-photosensitizer conjugates remain in the cell suspension,
the switching valve 8 is controlled such that the culture medium is
fed to the analyzer 9, and the analyzer conducts an analysis (step
7). The analyzer 9 may include a liquid chromatograph mass
spectrometer, and an ELISA method may be used. When it is
determined based on the analysis result that the
antibody-photosensitizer conjugates or the ligands of the
antibody-photosensitizer conjugates remain, the process operation
in step 6 is performed again. The "remain" refers to the fact that
the antibody-photosensitizer conjugates or ligands of the
antibody-photosensitizer conjugates in the cell suspension, for
example, have a concentration that causes abnormal differentiation
of cultured cells (e.g. a concentration in the cell suspension of
10% or more). The abnormalities here include tumorigenesis
(teratoma, carcinogenesis), abnormal differentiation, chromosomal
abnormalities, cell death, etc.
[0090] When it is determined from the analysis result obtained in
step 7 that the antibody-photosensitizer conjugates and the ligands
of the antibody-photosensitizer conjugates do not remain in the
cell suspension, the switching valve 8 and the liquid feeding pump
5 are controlled such that the cell suspension is fed to the cell
incubator 4 (step 8).
Embodiment 2: Batch
[0091] The present invention is applicable not only to the
flow-through purification device described above but also to a
so-called batch purification device.
[0092] An example of a batch purification device is shown in FIG.
3.
[0093] This embodiment includes a sample plate 12 on which a
plurality of cell culture vessels S are placed, a laser irradiator
11 that emits a laser beam, and a reflecting mirror 16 that
reflects the laser beam and concentrates it on the cell culture
vessels S placed on the sample plate 12. Observation images of the
cell culture vessels S are taken into an imaging unit 15 such as a
CCD camera via a reflecting mirror 17 and a condenser lens 18, and
image data corresponding to the observation images reproduced by
the imaging unit 15 is input to a control/processing unit 20.
[0094] The cell culture vessels S each contain a cell suspension
containing a culture medium and cells, and antibody-photosensitizer
conjugates. As an antibody photosensitizer, an antibody that
selectively binds to a cell to be killed, such as a conjugate
(Tra-IR700) of Tra (trastuzumab) and IR700 can be used.
[0095] The cell culture vessels S and the sample plate 12 are
preferably placed in a CO.sub.2 incubator (not shown). The CO.sub.2
incubator is a well-known one capable of adjusting the CO.sub.2
concentration and temperature of the atmosphere inside the
incubator, and plays a role in maintaining the cell culture
environment during the laser irradiation treatment, such as the pH
of the culture medium filled in each of the cell culture vessels S,
in a suitable state.
[0096] The control/processing unit 20 moves the sample plate 2 via
an XY drive unit 13 such that each cell culture vessel S comes to a
position at which the imaging unit 15 can capture an image. When
the cell culture vessel S comes to the position at which it can be
imaged, the imaging unit 15 acquires an image of the entire vessel
and sends the image data to the control/processing unit 20. The
control/processing unit 20 obtains a brightness value for each
pixel of the acquired image. Then, using the obtained brightness
values of all the pixels, a brightness value histogram showing the
relationship between the brightness values and the frequency of
occurrence of the pixels is created, and whether or not it is a
cell region and whether or not it is a cell to which the antibody
photosensitizer has bound are identified.
[0097] After the cell region is identified, a laser beam is
radiated from the laser irradiator 11 toward the cell culture
vessels S such that the antibody-photosensitizer conjugates that
have bound to the cells to be killed in the cell culture vessels S
act as death switches, and only specific cells that have bound to
the antibodies can be killed.
[0098] The control/processing unit determines the presence or
absence of dead cells based on the cell image captured by the
imaging unit 15, and continues to radiate the laser beam from the
laser irradiator when the cells to be removed are not killed.
Other Embodiments
[0099] The present invention is not limited to the above
embodiments, but an irradiated layer containing
antibody-photosensitizer conjugates in a layer containing a
material that receives and absorbs a laser beam may be provided on
a cell culture vessel main body, such as a lid or a side surface of
the vessel, for example. According to this, among cell colonies on
the cell culture vessel, only unnecessary cells and cancer cells
that have not differentiated into desired cells can be killed in a
pinpointed manner, and a certain region in the irradiated layer of
the cell culture vessel is raster-scanned with a laser beam such
that all cells located in the region can be killed.
DESCRIPTION OF REFERENCE NUMERALS
[0100] 1: holder [0101] 2: detector [0102] 3: radiation light
source [0103] 4: cell incubator [0104] 5: liquid feeding pump
[0105] 6: introduction pump [0106] 7: conjugate storage [0107] 8:
switching valve [0108] 9: analyzer [0109] 10: control/processing
unit
* * * * *